Our long term goal is to elucidate the mechanisms of initiation and regulation of RNA polymerase II transcription. Our experimental approach is to reconstitute these processes with purified proteins from yeast, and bring the power of combined biochemical and genetic analysis to bear on the mechanisms. During the previous project period, we developed a fully defined, appropriately regulated transcription system. The sub cloning of genes for all components and expression of many of the proteins in recombinant form should enable the culmination of our efforts during the coming project period. Specific aims of the proposed research are as follows: 1. Reconstitution and functional analysis of TFIIH. 2. Reconstitution and functional analysis of mediator. 3. Characterization of mammalian mediator. 4. Reconstitution of yeast TAF-dependent transcription in vitro. 5. Studies of RSC-nucleosome interaction.